Despite the spectacular pace of molecular studies of opioids and their receptors, we do not yet know why opiates are analgesic -- that is, how do opiates selectively inhibit pain without affecting other sensations? Moreover, the puzzling ability of opiates to inhibit aching, persistent pain ("2nd pain") without inhibiting sharp, transient pain ("1st pain") is largely unaddressed in the literature, even though this underlies the clinical use of opiates for post-operative pain. The research in this proposal should explain the role of primary sensory neurons in the specificity of opiates for 2nd pain; that goal is possible because we are the first lab that can identify nociceptors in tissue culture. Nociceptors, the sensors for pain, are peripheral neurons that are specialized to detect noxious stimuli, stimuli so strong that they threaten or cause tissue damage. Because their cell bodies sit in ganglia along with neurons that sense the various other somatic sensations, nociceptors could never be identified in primary tissue culture. We have solved this problem, creating a primary tissue culture system that contains fluorescently labelled nociceptors along with other, un=labelled sensory neurons. The novel system should propel several avenues of research, including he role of nociceptors in the specificity of opiates for pain. Our research focuses on the inhibition of Ca2+ channels by the mu opioid receptor, the binding site for morphine, because Ca2+ channel inhibition directly suppresses neurotransmitter release from presynaptic terminals. Such presynaptic inhibition is widely recognized as a key element of opioid action. Methods will include patch clamp recording of Ca2+ channel activity, single cell PCR for opioid receptors, and antisense oligonucleotide suppression of genes for G protein subunits. Our most relevant prior research convinces us; (1) that we can distinguish nociceptors that subserve 1st pain from those that subserve 2nd pain and; (2) that we know the key functional properties of the signalling path between mu receptors and Ca2+ channels. The proposed experiments target three working hypotheses: (1) Ca2+ channels on non-nonciceptive sensory neurons are not inhibited by opioids; (2) Selective opiate suppression of Ca2+ channels on small, unmyelinated nociceptors contributes to selective opiate inhibition of 2nd pain; (3) Different subtypes of G proteins couple different presynaptic inhibitors to Ca2+ channels in nociceptors. Correlations between our data and existing clinical and psychophysical observations may suggest novel strategies for analgesia by explaining the basis of known analgesics. By fully describing the molecular details of the signal linking mu receptors to Ca2+channels, we expect to contribute to the search for biochemical differences between acute and chronic actions of opiates.